The teachings of all of the references cited herein are incorporated in their entirety herein by reference.
The present invention relates to the method of obtaining a plasmid contained in a difficult to separate heterogeneous plasmid DNA fraction, particularly one containing plasmids of similar sizes. Another aspect of the present invention are new plasmids obtained using the method according to the present invention, their derivatives and fragments, and the application of these products in biotechnology and medicine, particularly in gene therapy.
In modern biotechnology, we assume that a DNA fragment (gene) we introduce into a cell, eg. Escherichia coli, will be expressed, but an unprotected foreign DNA fragment would shortly be degraded into nucleotides. DNA fragments coding sequences of biologically significant proteins such as insulin, interferon or growth hormone are introduced into host cells via a vector. Vectors are DNA molecules which are able to replicate autonomously in certain types of cells, and which ensure the amplification of the introduced DNA fragment, and in many cases the efficient expression of genes.
Most often, vectors are derivatives of naturally occurring plasmids. However, to turn the latter into useful plasmid vectors, a series of modifications need to be introduced into them. It is necessary to equip them with a marker, such as a gene or genes responsible for easily discerned phenotypic characteristics such as antibiotic resistance, and also to maximally reduce its molecular mass (the smaller the vector, the larger its “capacity” and ease of manipulation). It is also necessary to introduce a single restriction site of a given type which will be used for cloning, or to remove excess ones (two or more).
The nucleotide sequence should be known in its entirety, so that it may be discerned whether it contains known, or related, genes which may pose a threat to the health or even lives of people, plants or animals. Knowing the sequence also allows one to remove undesirable nucleotide sequences or add desirable ones such as immunogenic sequences in DNA vaccines. It has been determined that plasmid DNA causes the strongest immune response when CG sequences abut two purine bases (adenine or guanine) at the “C” side and two pyrimidines (thymine or cytosine) on the “G” side as described by Roman et al., Nat. Med. 8:849-854, schematically represented by RRCGYY, and herewith indicated as imm1. A particular case of this sequence which has a particularly strong immunogenic effect is imm2, which has two thymine bases at the “G” end.
Furthermore, CG units in bacterial plasmids are not methylated, whereas in vertebrates they usually are. It has been hypothesized that vertebrate organisms recognize a large frequency of unmethylated CG units as a danger signal, hence the amplified immune response. In gene therapy such an amplified immune response is undesirable, because it may lead to the destruction of the therapeutic protein and the plasmid vector. This is why it is preferable to use plasmids with low GC pair content in gene therapy.
In biotechnology, the most useful vectors are the so-called expression vectors, which facilitate efficient synthesis of the proteins encoded by genes contained on the vector. Such vectors bear promoter sequences which facilitate transcription and translation, and sequences ensuring the stability of the synthesized protein. There are expression vectors known under the control of strong promoters, whose synthesis can lead to accumulations of a given protein totalling 30% or even more of total cellular protein. Such vectors have been used for years in the production of many well known and useful proteins, particularly ones with desirable pharmacological properties.
It is known that certain compound segments of DNA, called transposons, are able to place themselves in many portions of the host genome. This means that certain large DNA segments, known as insertion sequences (IS's), can, if they are located nearby, transpose themselves as a larger unit encompassing genes located between them. Complex units of this type form the transposon. They are found in Prokaryota, such as bacteria, but also in Eukaryota. 
Recently, many useful bacterial transposons have been discovered and characterised, among them one called Tn5. It is a 5.8 kilobase pair (kbp) segment of bacterial DNA, which can undergo insertion in many places in the chromosome, in plasmids, as well as in “latent” phages of gram-negative bacteria. It codes a bacterial resistance gene to aminoglycoside antibiotics, kanamycin and neomycin, as well as gentamycin resistance (G418) in eukaryotic cells. A restriction map of Tn5 was presented by Berg et al., Genetics 105, 813-828 (1983). Further information regarding the technology of Tn5 is contained in the review articles according to Berg and Berg, Bio/Technology 1, 417-435 (1983); and Berg and Berg, in Neidhardt et al., (ed.), “Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” ASM, Washington, D.C., Chapter 63, p. 1,071-1,109 (1987).
Starting with naturally occurring transposons, a series of artificial constructs have been made, meant for use in genetic engineering. For example, patent description U.S. Pat. No. 5,137,829 presents a new DNA transposone derived from transposone Tn5 useful in the production of mutants and rapid screening of long DNA sequences. The transposone described encompasses a partial Tn5 transposone sequence with oligonucleotide SP6 and T7 phage primers, conveniently placed near opposite ends of the Tn5 transposone in question.
Classical methods used to isolate plasmid DNA, commonly used in laboratory practice are usually based on electrophoretic techniques. Despite their many pluses, which made them so popular, these techniques also do have certain limitations, particularly concerning their resolution. They are not suitable for separating DNA fragments of similar sizes, especially when the mixture being separated contains decidedly varied amounts of the individual fragments. In this case, both fragments migrate through the electrophoretic gel as one band, and the presence of the less numerous fragment is masked by the more numerous fragment.
In the light of the above described in the state of the art, several technical problems may objectively be described, which are still awaiting resolution. It is worthwhile then to seek out natural plasmids with specific and useful properties from bacterial strains isolated from various sources. To realise this goal, it is desirable to obtain an easy method of isolating a plasmid contained in a heterogeneous, not easily separable fraction of plasmid DNA, particularly one containing various plasmids of similar sizes. It is particularly desirable to obtain new plasmids which could be used to produce new constructs useful in biotechnology, especially ones facilitating stable or regulated expression of desired proteins. In this context it is particularly desirable to obtain autonomic functional elements which could be used in the production of other, useful constructs. For example, it is still desirable to produce transcription regulatory elements, like strong transcriptional promoters.
Furthermore, it is also desirable to obtain new plasmids, which would contain a decreased number of immunogenic sequences. Such plasmids could be a convenient source of various constructs designed for medical use, particularly gene therapy.
The above described problems have been solved in the present invention.